EP1465996B1 - Composes de liaison raf/ras - Google Patents

Composes de liaison raf/ras Download PDF

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EP1465996B1
EP1465996B1 EP02793099A EP02793099A EP1465996B1 EP 1465996 B1 EP1465996 B1 EP 1465996B1 EP 02793099 A EP02793099 A EP 02793099A EP 02793099 A EP02793099 A EP 02793099A EP 1465996 B1 EP1465996 B1 EP 1465996B1
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gilz
raf
seq
peptide
protein
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EP1465996A2 (fr
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Carlo Riccardi
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Merck Serono SA
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Laboratoires Serono SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention concerns the activities of GILZ protein and of GILZ protein-derived compounds in the field of signal transduction.
  • GCHs Glucocorticoid Hormones
  • the efficacy of Glucocorticoid Hormones (GCHs) as therapeutic agents for many acute/chronic inflammatory and autoimmune diseases is, at least partly, due to the effect of GCHs on T cell development and function.
  • the properties of these cells are regulated by a number of stimuli having direct or indirect consequences on the coordinated expression of a number of genes involved in activation and clonal expansion, such as interleukin-2/interleukin-2 receptor.
  • Thymic epithelial cells produce GCHs and it has been proposed that these locally produced glucocorticoids participate in antigen-specific thymocyte development by inhibiting activation-induced gene transcription (Ashwell JD et al., 2000).
  • GCHs induce apoptosis in thymocytes and activated mature T cells, possibly helping the elimination of developing lymphocytes that are differentiating improperly including neoplastic lymphocytes (Ramdas J and Harmon JM, 1998). Paradoxically, GCHs may also promote survival of thymocytes (Vacchio MS et al., 1994) and, in the periphery, they inhibits the apoptosis induced by continuously antigen-stimulated T lymphocytes, (Activation Induced Cell Death, AICD).
  • GCHs such important regulators of T-cell development
  • many investigators are trying to identify genes whose expression is strictly regulated by GCHs.
  • These researches should provide alternative means to regulate the molecular mechanisms of T cells, overcoming at same time the limitations and the unwanted effects of GCHs, studying for example the effects on transcriptional activity in cells treated with dexamethasone (DEX) a very stable and potent GCHs analogue commonly used in experimental models (Feng A et al., 1995).
  • DEX dexamethasone
  • GILZ Glucocorticoid-Induced Leucine Zipper
  • Mouse GILZ (mGILZ, SWISSPROT Acc. No. Q9Z2S7), which was initially cloned by comparing mRNA species expressed in DEX-treated and untreated murine thymocytes, is encoded by an mRNA of 1972 nucleotides, with the open reading frame starting at position 206, and contains 137 amino acids.
  • Human GILZ (hGILZ, SWISSPROT Acc. No. Q99576), which has been cloned by homology with mGILZ, is encoded by an mRNA of 1946 nucleotides, with the coding sequence starting at position 241, and contains 134 amino acids.
  • GILZ protein sequence is homologous with other members of the Leucine zipper family, in particular DIP (Sillard R et al., 1993; Vogel P et al., 1996), TSC-22 (Jay P et al., 1996; Shibanuma M et al., 1992) and thg-1 (Fiorenza MT et al., 2001).
  • DIP Leucine zipper family
  • TSC-22 Jay P et al., 1996; Shibanuma M et al., 1992
  • thg-1 Fuorenza MT et al., 2001
  • GILZ and GILZ-like proteins have been disclosed elsewhere, and described under other names, for example HT22L proteins ( WO 98/50425 ) or SEQ ID NO: 35 ( WO 00/77255 ), and present a central domain containing the Leucine zipper allowing the dimerization, which divides the N-terminal protein domain from the proline-rich C-terminal domain.
  • GILZ has been found expressed in normal T lymphocytes in the thymus, spleen, and lymph nodes, splenic B cells, and peritoneal macrophages. GILZ gene expression is strongly upregulated by DEX treatment in all those cells. In particular, GILZ is up-regulated by DEX treatment in mouse and human T lymphocytes and is down-regulated by treatment with ⁇ -CD3 antibody (in mouse cells) or phytohemagglutinin (in human cells). These results indicate that T-cell activation decreases GILZ expression and suggest that the two events (GILZ expression and T cell activation) might be mutually exclusive (Riccardi C et al., 2000).
  • GILZ As evaluated by Northern blot analysis, there are also non-lymphoid tissues poorly expressing GILZ are brain, kidney and liver. Recently, various articles described the altered expression of GILZ in different cells and tissues, such as primary osteosarcoma cells (Khanna C et al., 2001) or shear stressed human umbilical vein endothelial cells (McCormick SM et al., 2001).
  • GILZ over-expressing cells show that this protein is able to move into the nucleus and to protect T cells from TCR-activated apoptosis, but not from other apoptotic stimuli, mimicking the functional unresponsiveness and other effects of GCHs that have been described involved in the GCH-mediated immunosuppressive and antiinflammatory activity.
  • This anti-apoptotic effect correlates with the inhibition of activation-induced Fas/FasL and interleukin-2/interleukin-2 receptor up-regulation, has been associated to the GILZ property of binding NF-kB, blocking consequently the nuclear translocation and DNA binding of the NF-kB subunits, without affecting I-kB phosphorylation and degradation or I-kB/NF-kB binding (Ayroldi E et al., 2001).
  • GILZ GILZ expression inhibits the induction of reporter constructs driven by the FasL, AP-1, NF-AT, or IL-2 promoters (Mittelstadt PR and Ashwell JD, 2001).
  • GILZ was shown capable to interact with c-Fos and c-Jun, inhibiting the NFAT/AP-1- driven transcription. Both c-Fos and c-Jun were efficiently retained by the N-terminal portion of GILZ (residues 1-60), which lacks the leucine zipper, while the C-terminal portion of GILZ (residues 61-137) retained the ability to homodimerize, even though it is not excluded the possibility of heterodimerazation with other Leucine zipper proteins.
  • Raf-1 is a proto-oncogene belonging to a family of Serine/Threonine kinases, to which A-Raf and B-Raf also belong, having distinct tissue distribution and regulation.
  • Raf kinases can phosphorilate and activate, with different efficacy, the Mitogen activated/Extracellular regulated kinases 1 and 2 (MEK-1/-2), which in turn activate mitogen-activated protein kinase (MAPKs) and extracellular signal-regulated kinases (ERKs), leading to the propagation of the signal.
  • MEK-1/-2 Mitogen activated/Extracellular regulated kinases 1 and 2
  • MAPKs mitogen-activated protein kinase
  • ERKs extracellular signal-regulated kinases
  • the Raf-MEK-ERK cascade regulates diverse cellular processes such as proliferation, differentiation, and apoptosis.
  • Raf was initially identified as a protein reversibly interacting with Ras, a small GTP-binding protein and well studied proto-oncogene.
  • the activation of Ras initiates a complex array of signal transduction events, typical of higher eukaryotes and initiated by receptor and non-receptor tyrosine kinases requiring Raf in order to transduce growth and differentiation signals.
  • Raf is the Ras substrate and effector best characterized so far, and this kinase is considered as a central component in the signaling pathways involved in normal cell growth and differentiation. Active Ras stimulates, through MAPKs pathway, the phosphorylation and activation of ELK-1 that, in turn, induces transcription of c-Fos and JunB genes. The regulation of Raf activity appears very complex due to the high number of Raf interactions identified so far.
  • a survey of the literature on Raf-associated and/or Raf-affecting molecules reveals several categories of molecules possibly interacting with Raf, including G-proteins, adaptors, chaperons, phosphatases, receptors, kinases, phospholipids (Kolch W, 2000).
  • Raf and the MAPKs pathway play a fundamental role in the T cell growth and differentiation (Rincon M, 2001), but many evidences suggest that their action is coordinated with the action of GCHs.
  • Glucocorticoid Receptor can repress transactivation to AP-1 and NF-kB without the binding of GR to DNA (De Bosscher K et al., 1997), but GR and Raf can be found as well within the same protein complex (Widen C et al., 2000). Therefore, GR/Raf interaction may be responsible of inhibition of MAPK pathway, which can be achieved also with low concentrations of DEX (Rider LG et al., 1996).
  • Ras is locked in its GTP-bound form as a consequence of mutations, leading to constitutive signaling.
  • the Ras pathway no longer requires an upstream growth signal, and Ras downstream components, such as Raf and ERK-1/-2, are constitutively activated.
  • This process causes cell transforming phenotypes, such as lost of contact growth inhibition, growing in semi-solid medium, and increased proliferation rate.
  • Abnormal expression and/or mutations of Ras and Raf have been shown to trigger these transformed phenotypes caused by the interaction of Ras with Raf.
  • Means to modulate Raf activity and interactions may allow a control on the downstream signal transduction pathway that induces proliferation or differentiation to treat cancers or inhibit metastasis (Kloog Y and Cox AD, 2000).
  • Raf interactions in particular with Ras, have been intensively studied to elucidate the binding determinants and the functional consequences.
  • the aim of these researches is to help the development of molecules, directed either to Ras or to Raf, potentially useful in cancer therapeutics.
  • various mutants and peptides, derived from Ras or Raf, or obtained from computational and structural methods, have been disclosed in the literature as being inhibitor of the Ras/Raf interactions and / or signaling activities ( WO 97/34146 ; Barnard D et al., 1998; Zeng J et al., 2001; Williams JG et al., 2000; Maruta H et al., 2002; Ohnishi M et al., 1998; Winkler DG et al., 1998; Radziwill G et al., 1996; Block C et al., 1996).
  • Raf/Ras activated signaling pathway is deeply involved in control of cell proliferation and oncogenic transformation, it would be desirable to identify physiologically active molecules binding Raf/Ras and inhibiting this activation.
  • Such Raf/Ras interacting agents could be administered to a human or veterinary patient in a pharmaceutically acceptable form and in a therapeutically effective dosage for prophylaxis and therapy of pathological conditions related to elevated or prolonged Raf/Ras mediated signaling activity.
  • GILZ protein interacts directly with Raf/Ras complex, inhibiting the activation of the signaling pathway controlled by such complex. More specifically, it has now been found that a specific segment in the N-terminal region of GILZ interacts with Raf. These evidences can be exploited to use GILZ, and more particularly N-terminal segments of GILZ, as well as peptides and other molecules designed on the sequence and the structure of the N-terminal domain of GILZ protein.
  • GILZ can also affect the Raf/Ras complex-mediated intracellular signaling cascade by the means of a protein-protein interaction. This interaction could be responsible, at least in part, for GILZ-induced TCR unresponsiveness and, via inhibition of Raf-MEK-ERK activation pathway, for the regulation of the immune response mediated by GCHs.
  • the present invention provides the use of GILZ protein for inhibiting the Mitogen Activated Protein Kinases (MAPKs) pathway in an organism, in an organ, in a tissue, or in cultured cells.
  • MAPKs Mitogen Activated Protein Kinases
  • novel peptides capable of binding Raf protein and of inhibiting the MAPKs pathway are selected from:
  • amino acid sequence SEQ ID NO: 3 corresponds to the residues 16-36 of mouse GILZ, which has been shown to be the region of GILZ which binds Raf, mediating the inhibition of MAPKs cascade determined by GILZ, in the examples of the present patent application.
  • the polypeptides or peptides comprising at least 5 consecutive amino acids of SEQ ID NO: 3 are fragments of the N-terminal domain of GILZ corresponding to structural elements of such region, as well GILZ fragments including, partly or completely, sequences belonging to one or more of these structural elements.
  • these peptides correspond to the sequences GILZ(1-20), GILZ(21-50), GILZ (1-50), GILZ (10-30), GILZ (10-40), GILZ (16-22), GILZ (30-36), GILZ (10-50), GILZ (30-50), GILZ(16-58), or GILZ(1-36).
  • Such fragments are essentially GILZ "analogs", that is, displaying substantially the same novel biological activity of GILZ characterized in the present invention, as determined by means of routine experimentation comprising subjecting such an analog to the assays disclosed in the Examples below.
  • These analogs are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable thereof.
  • Deletions, substitutions, or additions in the above defined GILZ fragments can provide novel molecules which are active mutants of such fragments.
  • Active mutants of the polypeptide or peptide as defined in the present invention, or nucleic acid coding therefore, include a finite set of substantially corresponding sequences as substitution peptides or polypeptides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and functional features presented in the Examples. Nonetheless, they should display the same biological activity (i.e. inhibiton of Raf/Ras complex mediated signal transduction) as demonstrated in the present invention, or by any other relevant means known in the art, at comparable or higher levels.
  • preferred changes in these active mutants are commonly known as “conservative” or “safe” substitutions.
  • Conservative amino acid substitutions are those with amino acids having sufficiently similar chemical properties, in order to preserve the structure and the biological function of the molecule. It is clear that insertions and deletions of amino acids may also be made in the above defined sequences without altering their function, particularly if the insertions or deletions only involve a few amino acids, e.g., under thirty, and preferably under ten, and do not remove or displace amino acids which are critical to the functional conformation of the relevant GILZ fragment.
  • the literature provide many models on which the selection of conservative amino acids substitutions can be performed on the basis of statistical and physico-chemical studies on the sequence and/or the structure of natural protein (Rogov Sl and Nekrasov AN, 2001). Protein design experiments have shown that the use of specific subsets of amino acids can produce foldable and active proteins, helping in the classification of amino acid substitutions which can be more easily accommodated in protein structure, and which can be used to detect functional and structural homologs and paralogs (Murphy LR et al., 2000).
  • the synonymous amino acid groups and more preferred synonymous groups are those defined in Table I.
  • active mutants may be designed for improving certain properties independent from Raf/Ras interaction.
  • active mutants may result from the introduction of metal binding site(s) for improving protein stability, without loss of function, in particular by replacing surface residues in a loop / turn region with histidine capable of binding His-metal ligands, such as nickel cation (Bell AJ Jr et al., 2002).
  • Identity refers to the subunit sequence similarity between two polymeric molecules, e.g., two peptides. When a subunit position in both of the two molecules is occupied by the same monomeric unit, e.g., if a position in each of two peptides is occupied by Serine, then they are identical at that position.
  • the identity between two sequences is a direct function of the number of matching or identical positions, e.g., if identical; if 90% of the positions, e.g., 9 of 10, are matched, the two sequences share 90% sequence identity.
  • polypeptide is used herein as a generic term to refer to native or recombinant proteins, fragments, or analogs of a polypeptide sequence.
  • native or recombinant proteins, fragments, and analogs are species of the polypeptide group.
  • peptide will ordinarily applied to a polypeptidic chain containing from 4 to 80 or more contiguous amino acids, usually about 4-20 contiguous amino acids. Such peptides can be generated by methods known to those skilled in the art, including partial proteolytic cleavage of the protein, chemical synthesis of the fragment, or genetic engineering.
  • fragment refers to a polypeptide that has an N-terminal and/or C-terminal deletion when compared to the parent sequence (i.e. GILZ N-terminal region), but where the remaining amino acid sequence is identical to the corresponding positions in the naturally occurring sequence deduced, for example, from a full length cDNA sequence. Fragments typically contain at least 10 amino acids, preferably at least 20 amino acids or more.
  • active means that such alternative compounds should maintain the functional features characterized for GILZ and the specific fragment GILZ (18-36), accordingly to the present invention, and should be as well pharmaceutically acceptable, i.e. without imparting toxicity to the pharmaceutical compositions containing them.
  • polypeptides or peptides of the invention comprise the above defined GILZ fragments or their active mutants, and an amino acid sequence belonging to a protein other than GILZ;).
  • the peptides contain 4 to 25 amino acids, and have at least 70, 80, or 90% sequence identity to SEQ ID NO: 3.
  • the previous embodiments include, amongst the compounds of the invention, the amino acid sequence of other proteins belonging to the TSC-22/DIP family expressed by different organisms.
  • An example is human GILZ which differs from mouse GILZ only for one residue in position 22, a (Threonine instead of an Isoleucine; figure 12 ).
  • the present definition of the compounds of the invention comprises also the corresponding "fusion proteins", i.e. polypeptides comprising the amino acid sequence SEQ ID NO: 3, or fragments and active mutants thereof, and an amino acid sequence belonging to a protein other than any GILZ-like protein. This latter sequence may provide additional properties without impairing considerably functional binding and inhibiting activities.
  • polypeptides and the peptides of the present invention can provided in other alternative forms which can be preferred according to the desired method of use and/or production, for example as active fractions, precursors, salts, or derivatives.
  • fraction refers to any fragment of the polypeptidic chain of the compound itself, alone or in combination with related molecules or residues bound to it, for example residues of sugars or phosphates, or aggregates of the original polypeptide or peptide.
  • Such molecules can result also from other modifications which do not normally alter primary sequence, for example in vivo or in vitro chemical derivatization of peptides (acetylation or carboxylation), those made by modifying the pattern of glycosylation (by exposing the peptide to enzymes which affect glycosylation e.g., mammalian glycosylating or deglycosylating enzymes) or phosphorylation (introduction of phosphotyrosine, phosphoserine, or phosphothreonine residues) of a peptide during its synthesis and processing or in further processing steps.
  • the nature, the effect and the distribution of protein glycosylation have been reviewed in the literature (, 2002; Thanka Christlet TH and Veluraja K, 2001; Imperial
  • the "precursors” are compounds which can be converted into the compounds of present invention by metabolic and enzymatic processing prior or after the administration to the cells or to the body.
  • salts herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the peptides, polypeptides, or analogs thereof, of the present invention.
  • Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like.
  • Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.
  • mineral acids such as, for example, hydrochloric acid or sulfuric acid
  • organic acids such as, for example, acetic acid or oxalic acid.
  • any such salts must have substantially similar activity to the peptides, polypeptides of the invention or its analogs.
  • derivatives refers to derivatives which can be prepared from the functional groups present on the lateral chains of the amino acid moieties or on the terminal N- or C- groups according to known methods.
  • Such derivatives include for example esters or aliphatic amides of the carboxyl-groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl-groups and are formed with acyl-groups as for example alcanoyl- or aroyl-groups.
  • useful conjugates or complexes of the antagonists of the present invention can be generated as derivatives, using molecules and methods known in the art for improving the detection of the interaction with other proteins (radioactive or fluorescent labels, biotin), therapeutic efficacy (cytotoxic agents, isotopes), or drug delivery efficacy, such as polyethylene glycol and other natural or synthetic polymers (Pillai O and Panchagnula R, 2001).
  • the antagonists may be produced following a site-directed modification of an appropriate residue, present in the natural sequence or introduced by mutating the natural sequence, at an internal or terminal position. Similar modifications have been already disclosed for small polypeptides such as chemokines ( WO 02/04499 ; WO 02/04015 ; Vita C et al., 2002).
  • Any residue can be used for attachment, provided it has a side-chain amenable for polymer attachment (i.e., the side chain of an amino acid bearing a functional group, e.g., lysine, aspartic acid, glutamic acid, cysteine, histidine, etc.).
  • a residue at these sites can be replaced with a different amino acid having a side chain amenable for polymer attachment.
  • the side chains of the genetically encoded amino acids can be chemically modified for polymer attachment, or unnatural amino acids with appropriate side chain functional groups can be employed.
  • Polymer attachment may be not only to the side chain of the amino acid naturally occurring in a specific position of the antagonist or to the side chain of a natural or unnatural amino acid that replaces the amino acid naturally occurring in a specific position of the antagonist, but also to a carbohydrate or other moiety that is attached to the side chain of the amino acid at the target position.
  • Polymers suitable for these purposes are biocompatible, namely, they are nontoxic to biological systems, and many such polymers are known.
  • Such polymers may be hydrophobic or hydrophilic in nature, biodegradable, non-biodegradable, or a combination thereof.
  • These polymers include natural polymers (such as collagen, gelatin, cellulose, hyaluronic acid), as well as synthetic polymers (such as polyesters, polyorthoesters, polyanhydrides).
  • hydrophobic non-degradable polymers include polydimethyl siloxanes, polyurethanes, polytetrafluoroethylenes, polyethylenes, polyvinyl chlorides, and polymethyl methaerylates.
  • hydrophilic non-degradable polymers examples include poly(2-hydroxyethyl methacrylate), polyvinyl alcohol, poly(N-vinyl pyrrolidone), polyalkylenes, polyacrylamide, and copolymers thereof.
  • Preferred polymers comprise as a sequential repeat unit ethylene oxide, such as polyethylene glycol (PEG).
  • the preferred method of attachment employs a combination of peptide synthesis and chemical ligation.
  • the attachment of a water-soluble polymer will be through a biodegradable linker, especially at the amino-terminal region of a protein.
  • Such modification acts to provide the protein in a "pro-drug" form, that, upon degradation of the linker releases the protein without polymer modification.
  • the invention includes alternative molecules based on GILZ-derived peptides which are generated in the form of peptide mimetics (also called peptidomimetics), that is, GILZ analogs in which the nature of peptide or polypeptide has been chemically modified at the level of amino acid side chains, of amino acid chirality, and/or of the peptide backbone. These alterations are intended to provide GILZ agonist compounds having similar or improved therapeutic and/or pharmacokinetic properties.
  • peptide when the peptide is susceptible to cleavage by peptidases following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a non-cleavable peptide mimetic can provide a peptide more stable and thus more useful as a therapeutic.
  • replacement of an L-amino acid residue is a standard way of rendering the peptide less sensitive to proteolysis, and finally more similar to organic compounds other than peptides.
  • amino-terminal blocking groups such as t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl.
  • Methods for optimising the peptide structure of series of peptides derived from a scaffold, following a computational stabilization and/or optimization of proteins can be developed using cell-/ peptide-based microarrays or other experimental screening technologies ( WO 02/90985 ; Wu RZ et al., 2002; Filikov AV et al., 2002; Hayes RJ et al., 2002).
  • compounds of the invention having MAPKs cascade inhibiting properties and having as well some lipophilic characteristics may be most useful in view of the fact that in practice, such compounds to be used pharmaceutically should have the ability to pass through the cell membrane.
  • such compounds can be chemically modified (i.e. derivatized, conjugated or complexed) with molecules that, being transported naturally across the cell membrane, facilitate their entry or enhance their permeability across the cell membrane and into the cytoplasm.
  • these membrane blending agents are fusogenic polypeptides, ion-channel forming polypeptides, other membrane polypeptides, and long chain fatty acids, e.g., myristic acid, palmitic acid ( US 5149782 ).
  • These membranes blending agents insert the molecular conjugates into the lipid bilayer of cellular membranes and facilitate their entry into the cytoplasm.
  • Other valuable methods for transmembrane delivery of molecules exploit the mechanism of receptor mediated endocytotic activity.
  • These receptor systems include those recognizing galactose, mannose, mannose 6-phosphate, transferrin, asialoglycoprotein, transcobalamin (vitamin B 12), insulin and other peptide growth factors such as epidermal growth factor (EGF).
  • Nutrient receptors such as receptors for biotin and folate
  • Nutrient receptors can be also advantageously used to enhance transport across the cell membrane due to the location and multiplicity of biotin and folate receptors on the membrane surfaces of most cells and the associated receptor mediated transmembrane transport processes ( US 5108921 ).
  • a complex formed between a compound to be delivered into the cytoplasm and a ligand, such as biotin or folate can be contacted with a cell membrane bearing biotin or folate receptors to initiate the receptor mediated trans-membrane transport mechanism and thereby permit entry of the desired compound into the cell.
  • Specific examples for intracellular delivery of Ras/Raf interacting peptides are provided in the literature (Maruta H et al., 2002).
  • Peptides may be altered to increase lipophilicity (e.g. by esterification to a bulky lipophilic moiety such as cholesteryl) or to supply a cleavable "targetor" moiety that enhances retention on the brain side of the barrier (Bodor et al., 1992).
  • the peptide may be linked to an antibody specific for the transferrin receptor, in order to exploit that receptor's role in transporting iron across the blood- brain barrier ( Friden et al., Science 1993, 259: 373-377 ).
  • Other methods of biomimetic transport and rational drug delivery in the field of transvascular drug delivery are known in the art ( Ranney DF, Biochem Pharmacol 2000, 59: 105-14 ).
  • the compounds of the invention may be prepared by any well known procedure in the art, including recombinant DNA-related technologies or chemical synthesis technologies.
  • the expression of peptides and polypeptides of the invention can be achieved in an Eukaryotic or Prokaryotic cell by introducing an expression vector that, either integrated in the genome of the cell or maintained as an episome, contains the nucleotide sequence coding for the desired polypeptide or peptide under the control of transcriptional initiation/termination regulatory sequences which are constitutively active or inducible in said cell.
  • the relevant coding sequence may be already present in the genomic DNA of the cell and its expression can be activated by introducing exogenous regulatory sequences, as described in the prior art ( EP505500 ).
  • the DNA sequences coding for the GILZ-derived peptides and proteins of the invention can be isolated from the corresponding human or mouse genomic DNA or cDNA sequences, or any other nucleic acid sequences which, by virtue of the degeneracy of the genetic code, also encodes for the given amino acid sequences.
  • Expression vectors which comprise the above DNAs, together with any appropriate stop / start trascription and translation elements and any other additional sequence (e.g. heterologus sequence to be included in a fusion protein) can be used to transform host cells which can be cultured in an appropriate culture media, before collecting the expressed proteins and further processing.
  • Expression of any of the recombinant proteins of the invention as mentioned herein can be effected in Eukaryotic cells (e.g. yeasts, insect or mammalian cells) or Prokaryotic cells, using the appropriate expression vectors. Any method known in the art can be employed.
  • the coding sequences can be accordingly chosen in order to have an optimal codon usage for expression according to the specific the host cell, for example E. coli (Kane JF et al., 1995).
  • Recombinant proteins having the desired glycosylation pattern can be obtained by selecting the appropriate mammalian host cells (Grabenhorst E et al., 1999).
  • mammalian cells such as human, monkey, mouse, and Chinese hamster ovary (CHO) cells in particular, are preferred because they provide post-translational modifications to protein molecules, including correct folding or glycosylation at correct sites.
  • yeast cells can carry out post-translational peptide modifications including glycosylation.
  • Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides, signal sequences).
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector, may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • the vectors should allow the expression of the isolated or fusion protein including the antagonist of the invention in the Prokaryotic or Eukaryotic host cell under the control of transcriptional initiation / termination regulatory sequences, which are chosen to be constitutively active or inducible in said cell.
  • the host cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the desired proteins.
  • a cell line substantially enriched in such cells can be then isolated to provide a stable cell line.
  • Eukaryotic hosts e.g. yeasts, insect or mammalian cells
  • different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived form viral sources, such as adenovirus, bovine papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of the Herpes virus, the SV40 early promoter, the yeast ga14 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression and activation, so that expression of the genes can be modulated.
  • the cells which have been stably transformed by the introduced DNA can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may also provide for phototrophy to an auxotropic host, biocide resistance, e.g. antibiotics, or heavy metals such as copper, or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection.
  • the GILZ-derived peptides and proteins of the invention may be prepared by any other well known procedure in the art, in particular, by the chemical synthesis procedures, which can be efficiently applied on these molecule given the short length. Even totally synthetic proteins, also containing additional chemical groups, are disclosed in the literature (Brown A et al., 1996; Vita C et al., 2002).
  • Examples of chemical synthesis technologies are solid phase synthesis and liquid phase synthesis.
  • a solid phase synthesis for example, the amino acid corresponding to the C-terminus of the peptide to be synthesised is bound to a support which is insoluble in organic solvents, and by alternate repetition of reactions, one wherein amino acids with their -amino groups and side chain functional groups protected with appropriate protective groups are condensed one by one in order from the C-terminus to the N-terminus, and one where the amino acids bound to the resin or the protective group of the -amino groups of the peptides are released, the peptide chain is thus extended in this manner.
  • Solid phase synthesis methods are largely classified by the tBoc method and the Fmoc method, depending on the type of protective group used.
  • protective groups include tBoc (t-butoxycarbonyl), CI-Z (2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl (benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh (4,4'-dimethoxydibenzhydryl), Mtr (4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl), Z (benzyloxycarbonyl) and Cl2-Bzl (2,6-dichlorobenzyl) for the amino groups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl) for the guanidino groups); and tBu (t-buty
  • Such peptide cutting reaction may be carried with hydrogen fluoride or trifluoromethane sulfonic acid for the Boc method, and with TFA for the Fmoc method.
  • Purification of the synthetic or recombinant proteins may be carried out by any one of the methods known for this purpose, i.e. any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like.
  • HPLC high performance liquid chromatography
  • the elution can be carried using a water-acetonitrile-based solvent commonly employed for protein purification.
  • a further purification procedure that may be used in preference for purifying the peptides or proteins of the invention is affinity chromatography using monoclonal antibodies, heparin, or any other suitable ligand which can bind the target protein at high efficiency and can be immobilized on a gel matrix contained within a column. Impure preparations containing the proteins are passed through the column. The protein will be bound to the column by means of this ligand while the impurities will pass through. After washing, the protein is eluted from the gel by a change in pH or ionic strength.
  • the invention includes purified preparations of the compounds of the invention.
  • Purified preparations refers to the preparations which contain at least 1 %, preferably at least 5%, by dry weight of the compounds of the invention.
  • the GILZ-derived peptides and proteins of the present invention can be also used in methods and kits for screening in vitro and in vivo compounds possibly binding to Raf and/or Ras and inhibiting MAPKs pathway, as provided in the Examples of the present patent application, by comparing the effect of such compounds with the effect provided by the GILZ-derived peptides and proteins of the invention.
  • the components of this screening assay can be immobilized (by adsorption onto a plastic microtiter plate or specific binding of a fusion protein to a polymeric bead containing an affinity group), co-precipitated (by antibodies), and/or can be labeled (using radioisotopes, enzymatic labels, fluorescers, chemiluminescers).
  • the present invention also provides compounds isolated, identified and/or characterized by any of the above in vivo or in vitro assays and exemplified in the present patent application, as well any other chemical compound identified by methods of computer-aided drug design which make use of the sequence and structure information related to Raf/Ras and GILZ, in particular of the respective binding surfaces, to derive peptides or other organic compounds to be synthetized and tested in vitro and in vivo as inhibitors of MAPKs pathway.
  • Another aspect of the invention are methods for inhibiting unwanted cell proliferation mediated by the MAPKs in an animal, in an organ, in a tissue, or in cultured cells by administering an effective amount of a GILZ-derived compound of the invention.
  • These molecules can inhibit the cellular mechanisms triggered by the phosphorylation of Raf and Erk-1/-2, which play a crucial role in the transduction of signal from the cell membrane and cytoplasmatic receptors towards the transcriptional machinery in the nucleus.
  • the GILZ interaction provides the inhibition of the Raf/Ras-mediated intracellular signaling. Such inhibition is desirable in the treatment of unwanted cell proliferation, in general, and cancer, in particular.
  • Raf is a direct major effector of Ras function.
  • the requirement of Raf activity for Ras effector signalling allows the compounds of the present invention to interrupt the Ras protein pathway of oncogenic activation in tumor cells (Kloog Y and Cox AD, 2000; Weinstein-Oppenheimer CR et al., 2000).
  • the present invention provides a novel opportunity for the development of anticancer drugs targeting the MAP kinase pathway and controlling aberrant patterns of differentiation and proliferation (Sebolt-Leopold JS, 2000)
  • the interruption of the Raf/Ras-controlled MAPKs signaling cascade is expected to have antitumour activity in at least a proportion of human tumors (carcinomas, hematopoietic tumors of lymphoid/myeloid lineage, tumors of mesenchymal origin, melanoma). Therefore, the invention also relates to methods of manufacturing the compounds and pharmaceutical compositions, and methods of treating autoimmune or inflammatory diseases or cancers (such as lymphomas or lymphocytic leukaemia), which are triggered by Raf/Ras complex mediated activation.
  • autoimmune or inflammatory diseases or cancers such as lymphomas or lymphocytic leukaemia
  • GILZ can be used for the manufacture of a pharmaceutical composition for the treatment of autoimmune or inflammatory diseases or cancers. In such treatments, it can be sometimes advantageous to target the compounds of the present invention to the cancerous cells with the higher precision and specificity. Such targeting is well known within the art of cancer treatment and the preparation of suitable formulations and methods requires no more
  • this method may also be useful in downregulating the immune response in patients with inflammatory or autoimmune diseases such as systemic lupus erythematosus (SLE), type 1 diabetes, vasculitis, autoimmune chronic active hepatitis, ulcerative cholitiss, Crohn's disease, allergic diseases, nephritic syndrome, sarcoidosis, and rheumatoid arthritis. Suppression of an immune response using this method may also be useful in the treatment of allograft or xenograft recipients to prevent rejection of a transplanted organ.
  • SLE systemic lupus erythematosus
  • the therapeutic administration of a peptide intracellularly can also be accomplished using gene therapy, wherein a nucleic acid which includes a promoter operatively linked to a sequence encoding an heterologous polypeptide or peptide is used to generate high levels of expression in cells transfected with the previously described nucleic acid.
  • Plasmid DNA or isolated nucleic acid encoding GILZ-derived peptides or proteins of the invention may be introduced into cells of the patient by standard vectors and/or gene delivery systems. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, and adenoviruses, among others.
  • the compounds of the invention described above may thus be used as medicaments, in particular as the active ingredients in pharmaceutical compositions for the treatment of unwanted cell proliferation, in general, and cancer in particular.
  • Such treatments can be performed either in vivo, by administering the compound to the animal, or ex vivo, that is, the compounds are administered to an organ, a tissue, or cultured cells which have been extracted from the body and kept outside for a short period to provide a specific therapeutic treatment before being implanted again in the body.
  • the present invention also provides pharmaceutical compositions comprising one of the compounds of the invention, as active ingredient and a pharmaceutically acceptable carrier, excipient, stabilizer or diluent.
  • a pharmaceutically acceptable carrier excipient, stabilizer or diluent.
  • the composition or the isolated compounds of the invention can be administered alone or in combination with a another composition or compound which provide additional beneficial effects by acting in a synergic or in a coordinated manner.
  • therapeutically effective amount that is, an amount effective affect the course and the severity of the disease, leading to the reduction or remission of such pathology.
  • the effective amount will depend on the route of administration and the condition of the patient.
  • compositions may contain suitable pharmaceutically acceptable carriers, biologically compatible vehicles which are suitable for administration to an animal (for example, physiological saline) and eventually comprising auxiliaries (like excipients, stabilizers or diluents) which facilitate the processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers for example, biologically compatible vehicles which are suitable for administration to an animal (for example, physiological saline) and eventually comprising auxiliaries (like excipients, stabilizers or diluents) which facilitate the processing of the active compounds into preparations which can be used pharmaceutically.
  • auxiliaries like excipients, stabilizers or diluents
  • compositions may be formulated in any acceptable way to meet the needs of the mode of administration.
  • the use of biomaterials and other polymers for drug delivery, as well the different techniques and models to validate a specific mode of administration, are disclosed in literature (Luo B and Prestwich GD, 2001; Cleland JL et al., 2001).
  • administration may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, oral, or buccal routes.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art, and can be prepared according to routine methods.
  • suspension of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions that may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
  • Pharmaceutical compositions include suitable solutions for administration by injection, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active compound together with the excipient. Compositions which can be administered rectally include suppositories.
  • the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the pharmaceutical composition of the present invention may be administered alone or in conjunction with other therapeutics directed to the condition, or directed to other symptoms of the condition.
  • the compounds of the present invention may be administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline.
  • a pharmaceutically acceptable carrier such as physiological saline.
  • Standard methods for intracellular delivery of peptides can be used, e.g. delivery via liposomes. Such methods are well known to those of ordinary skill in the art.
  • the formulations of this invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight.
  • Ordinarily 1 to 40 milligrams per kilogram per day given in divided doses or in sustained release form is effective to obtain the desired results.
  • Second or subsequent administrations can be performed at a dosage, which is the same, less than, or greater than the initial or previous dose administered to the individual.
  • mouse GILZ cDNA coding sequence (414 base pairs; GenBank acc. n. AF024519) was cloned into a pcDNA3 plasmid (Invitrogen) for expression in 3DO cells.
  • Cells were transfected by electroporation (300 mA, 960 ⁇ F) with 15 ⁇ g linearized pcDNA3 vector (control clones) or 15 ⁇ g linearized pcDNA3 vector expressing the cDNA coding for mouse GILZ (pcDNA3-GILZ).
  • the plasmid expressing Myc-tagged GILZ was a pcDNA3.1/Myc-His vector (Invitrogen), containing the full-length mouse GILZ coding sequence which was PCR amplified and cloned between the BamHI and XbaI restriction sites.
  • Hamster monoclonal ⁇ -mouse CD3 ⁇ antibody (clone 145-2C11; Pharmingen) was diluted in phosphate-buffered saline (PBS) at 1 microgram/millilitre and distributed in flat-bottomed, high-binding 96 wells plates (Costar), putting 100 microliters for each well).
  • PBS phosphate-buffered saline
  • the antibodies were allowed to adhere at 4°C for 20 hours and, after being washed with PBS, the coated wells were used to plate the transfected clones (1 ⁇ 10 5 cells per well) and incubated at 37°C.
  • Nuclear cell extracts were prepared by resuspending 2X10 7 ice-cold PBS washed cells in 1 millilitre of hypotonic buffer containing HEPES (pH 7.5) 25 milliMolar, KCI 50 milliMolar, Nonidet P-40 (NP-40) 0.5%, dithiothreitol (DTT) 0.1 milliMolar, leupeptin 10 milligrams/millilitre, aprotinin 20 milligrams/millilitre, phenylmethylsulfonyl fluoride (PMSF) 1 milliMolar.
  • the cytoplasmic proteins-containing supernatants after 10 minutes of incubation on ice, were separated from nuclear pellets by centrifugation.
  • lysis buffer HEPES (pH 7.5) 25 milliMolar, KCI 2 milliMolar, DTT 0.1 milliMolar, leupeptin 10 milligrams/millilitre, aprotinin 20 milligrams/millilitre, PMSF 1 milliMolar.
  • the lysates obtained after 15 minutes of incubation on ice were diluted with 10 volumes of dilution buffer (HEPES (pH 7.5) 25 milliMolar, glycerol 20%, DTT 0.1 milliMolar, leupeptin 10 milligrams/millilitre, aprotinin 20 milligrams/millilitre, PMSF 1 milliMolar) and cleared in a precooled centrifuge for 30 minutes at 14000 RPM.
  • HEPES pH 7.5
  • GILZ primary antibodies were a rabbit polyclonal antiserum recognizing GILZ diluted 1:5000, or a monoclonal mouse ⁇ -human GILZ antibody prepared by immunizing BaIb/c mices with GST-human GILZ as antigen.
  • the antisera were first screened by ELISA using the antigen and positive spleen cells (cut-off dilution >1:800) were fused with myeloma cells I the presence of feeder cells.
  • Hybridoma supernatants were screened by ELISA and positive cells were cloned by limiting dilution, Some of them were used to inoculate mice intraperitoneally and obtain ascites fluid enriched in monoclonal antibodies. The ascites fluid was heat-inactivated, titered, and stored.
  • the secondary antibodies were horseradish peroxidase-labeled ⁇ -rabbit, ⁇ -rat, or ⁇ -mouse antibodies (depending on the primary antibody) provided by the SuperSignal chemiluminescence kit (Pierce), used according to manufacturer's instructions.
  • the antibodies against the non-phosphorylated protein variants were used to verify that no modulation of protein expression occurred, whilst ⁇ -beta tubulin antibody was used to check that an equivalent amount of proteins were loaded in each lane.
  • the primary and secondary antibodies previously used are "stripped" using the Restore Western Blot Stripping Buffer (Pierce), according to manufacturer's instructions.
  • Murine thymocytes were stimulated for 6 hours with DEX and then for different times with anti-CD3 monoclonal antibodies (from 30 minutes to 3 hours), obtaining similar results.
  • Cell extracts were tested by Western Blot as
  • the 3DO cells were transfected with the AP-1 luciferase reporter gene along pcDNA3 (control, empty vector) or pcDNA3-GILZ, with or without the cDNA of the activated form of Raf. Cloned in the pUSEamp vector (Upstate Biotechnology). Each transfection was performed by electroporation as above described in triplicate (5 micrograms of each plasmid). The transfection efficacy was assessed by co-transfeting a plasmid expressing Green Fluorescent Protein. Cell lysis and luciferase quantification were performed using commercial reagents (Roche Diagnostics).
  • the proportion of cells in the different cell cycle phases in the cell proliferation assay was evaluated by propidium iodide solution and flow cytometry. Briefly, cells were centrifuged and the pellets resuspended in 1.5 mL hypotonic propidium iodide (PI) solution. The tubes were kept at 4°C in the dark overnight. The PI-fluorescence of individual nuclei was measured by flow cytometry with standard FACScan equipment (Becton Dickinson). The cell cycle was analysed by Cell Fit program. Transfected clones were prepared as previously described by electroporation (3DO cells) or by using Lipofectamine (H 35 rat hepatoma cells) following manufacture's instruction (Gibco BRL), and analyzed after two days.
  • PI propidium iodide
  • Radiolabeled Thymidine incorporation assay was performed by culturing the cells for 24 hours at serial concentrations (from 1X10 5 to 0.175X10 5 per well). 2.5 ⁇ Ci [ 3 H]-thymidine per well were added 15 hours before harvesting with a multiple suction-filtration apparatus (Mash II) on a fiberglass filter (Whittaker Co.) and counted in a ⁇ counter (Packard).
  • GILZ overexpression inhibits c-Fos transcription.
  • GILZ specifically interacts in vitro with c-Fos and c-Jun in vitro, inhibiting the binding of active AP-1 to its target DNA (Mittellstadt PL et Ashwell JD, 2001). It was now addressed the possibility that GILZ could also interfere with the upstream AP-1 activation pathways, such as MAPKs activation and c-Fos and c-Jun transcription.
  • GILZ overexpression inhibits ERK-1 / -2 and Raf, but not JNK phosphorylation
  • c-Fos protein could explain, in part, the decrease in transactivation of multimerized AP-1, whose transactivation (through c-Fos transcription and c-Jun phosphorylation) is under the control of Ras/MAPKs pathway (Whitehurst CE and Geppert TD, 1996).
  • GILZ is inducible by DEX and correlates with the inhibition of anti-CD3 induced signalling in murine thymocytes, in particular, with the phosphorylation of Raf and of downstream proteins MEK and ERK-1/-2, which was reduced in a coordinated manner ( figure 3 ).
  • JNK which controls C-Jun phosphorylation and transcription and whose activation is under control of stress-activated MAPKs pathway, was already phosphorylated in non-stimulated 3DO clones (both empty- and GILZ transfected-clones).
  • the lack of modulation of JNK activation well matched with the lacked modulation of c-Jun transcription observed in the experiment showed in figure 1 .
  • the plasmid allowing the overexpression of GILZ was transfected in various cell types to verify if GILZ, in view of the previous experiments showing an effect on the signal transduction pathway controlling cell proliferation, has an effect on this cell function.
  • the data observed in some cell lines confirmed the anti proliferative activity of GILZ, which drives the accumulation of cells in phase G 0 G 1 of cell cycle (Table III). These evidences on the control of cell proliferation mediated by GILZ were also verified also by measuring the uptake of radiolabeled thymidine.
  • Example 2 mechanisms of the GILZ-mediated inhibition of the Raf-controlled MAPKs transduction pathway.
  • Immunoprecipitations were performed in RIPA buffer (TRIS (pH 7.5) 50 milliMolar, NaCl 150 milliMolar, Nonidet P-40 1%, deoxycholate 0.5%, sodium dodecyl sulphate (SDS) 0.1%, and EDTA 5 milliMolar) supplemented with 1 mM PMSF.
  • RIPA buffer TriS (pH 7.5) 50 milliMolar, NaCl 150 milliMolar, Nonidet P-40 1%, deoxycholate 0.5%, sodium dodecyl sulphate (SDS) 0.1%, and EDTA 5 milliMolar
  • ⁇ -Raf ⁇ -NF-AT
  • ⁇ -Ras antibodies Upstate Technology
  • Antigen-antibody complexes were precipitated with protein A-Sepharose beads (Pharmacia) and dissociated from beads prior to SDS-PAGE by boiling in loading buffer.
  • COS-7 lysates 500 ⁇ g were immunoprecipitated in RIPA buffer with ⁇ -Myc antibodies (4 ⁇ g/mg protein, Invitrogen) and western blot performed with ⁇ -Myc antibodies (1 ⁇ g/ml, Invitrogen,) or ⁇ -Raf antibodies (Upstate Biotechnology). The Western blots were performed as described above.
  • Glutathione S-transferase GILZ fusion protein (GST-GILZ) was prepared as previously described (Ayroldi E et al., 2001).
  • GST-Raf-RBD the GST fusion protein comprising the Ras Binding Domain of human Raf (Raf-RBD, residues 1-149; De Rooij J and Bos JL, 1997) was cloned in the same expression vector pGEX-4T2 plasmid (Pharmacia) and obtained in the same way.
  • Extracts were prepared from the indicated cell, treated or untreated with DEX (10 microMolar), as previously described (Ayroldi E et al., 2001).
  • GST or GST fusion proteins, loaded on Sepharose beads were mixed with cell extracts in binding buffer (NaCl 250 miliiMolar, HEPES (ph7.5) (pH 7.5) 50 milliMolar, EDTA 0.5 milliMolar, Nonidet P-40 0.1% (v/v), phenylmethylsulfonyl fluoride (PMSF) 0.2 milliMolar, dithiothreitol (DTT) 1 milliMolar, bovine serum albumin (BSA) 100 ⁇ g/ml), heated for 5 minutes at 42 °C, incubated for 2 hours at 4°C, washed extensively with binding buffer, resuspended in loading buffer and analysed by SDS-PAGE and Western blot as described above.
  • binding buffer NaCl 250 miliiMolar, HEPES (ph7.5
  • In vitro translated proteins were diluted with binding buffer (HEPES (pH 7.5) 25 milliMolar, glycerol 10%, NaCl 50 milliMolar, Nonidet P-40 0.05%, 1 mM DTT) and pre-cleared with glutathione beads for 45 minutes at 4°C.
  • binding buffer HEPES (pH 7.5) 25 milliMolar, glycerol 10%, NaCl 50 milliMolar, Nonidet P-40 0.05%, 1 mM DTT
  • GST or GST fusion proteins were bound to glutathione beads and incubated with in vitro translated proteins for 18 hours at 4°C.
  • the beads were subsequently washed five times with 0.5 millilitre of PBS and the proteins recovered by boiling the beads in SDS sample buffer were analysed by SDS-PAGE.
  • GILZ interacts with Raf and Ras.
  • Antigen-induced activation leads to conversion of Ras to its active form as well as activation of the kinase Raf. It has also been shown that treatment of mast cells with DEX blocked the phosphorylation of Raf, MEK, ERK-2 without affecting Ras activation (Cissel DS and Beaven MA, 2000). Since protein-protein interaction may have important consequences on protein phosphorylation, activation and trafficking, it has to be demonstrated if a DEX-induced protein such as GILZ is eventually capable of binding proteins belonging to the MAPKs cascade and to inhibit their activation.
  • GST-GILZ containing entire mouse GILZ
  • GST-GILZ was immobilised on beads and used in pull-down experiments with protein extracts obtained from DEX un-/stimulated 3DO cells, using beads loaded with GST as a control.
  • the proteins interacting with the beads were separated on a SDS-PAGE gel and transferred on a membrane then probed with an ⁇ -GILZ antibody.
  • a band immunoreactive with ⁇ -Raf antibodies is clearly detectable only when GST-GILZ is used in the pull-down experiment ( figure 6A ).
  • Murine thymocytes known to upregulate GILZ expression upon DEX stimulation.
  • Murine thymocytes were treated for 6 hours with DEX and the whole cell lysates were immunoprecipitated with antibodies recognizing either Raf or NF-AT.
  • the immunoprecipitated material was analyzed by Western blot using antibodies recognizing either GILZ or Raf.
  • the antibody against GILZ detects an immunoreactive protein only in the lysates immunoprecipitated with the ⁇ -Raf antibody and not with the ⁇ -NF-AT antibody.
  • a cell line (COS-7) was transfected with a plasmid expressing Raf with or without another plasmid expressing GILZ fused with Myc, an epitope helping the independent detection of GILZ.
  • the extracts obtained from the transfected cells were used in immunoprecipitation experiments where antibodies specific for Myc were applied.
  • the ⁇ -Myc antibody immunoprecipitates Raf in the whole cell lysates transfected with the plasmid expressing myc-GILZ only when the plasmid expressing Myc-GILZ is co-transfected.
  • the ⁇ -Myc antibody detects myc-GILZ protein in the whole lysate of cells transfected with myc-GILZ and Raf, as well as in material co-immunoprecipitated with ⁇ -Raf antibodies, but not in cell extracts obtained from cells transfected only with the plasmid expressing Raf ( figure 8 ).
  • similar results were obtained using antibodies against Ras in immunoprecipitation and Western blot.
  • Raf Ras binding domain of Raf (RBD, residues 51-131, Winkler DG et al., 1998) and was then called GST-Raf-RBD.
  • Glutathione S-transferase fusion protein including diffrent segments of GILZ were prepared by cloning the segment encoding for such GILZ fragments in the plasmid originally described for the expression of GST-GILZ (Ayroldi E et al., 2001). When necessary (i.e. whenever the original GILZ methionine was not included), a Met codon was added by at 5' of the sequence by normal genetic. The GST pull-down experiments were performed as described in the previous example with 3DO cells.
  • ⁇ C-GILZ contains the first 97 amino acids of mouse GILZ.
  • DN-GILZ contains the first 8 amino acids of mGILZ fused with the residues 73-137.
  • the in vitro translation with [ 35 S]-Methionine was performed with a commercial rabbit reticulocyte transcription-translation system (TNT, Promega).
  • GST-pull-down experiments were also performed using the recombinant GST-Raf-RBD fusion protein ( Figure 12 ).
  • GST-Raf-RBD fusion protein but not GST alone, binds GILZ full-length protein as well as the GILZ truncated form missing the C-terminal region ( ⁇ C-GILZ).
  • beads loaded with GST-Raf-RBD fusion protein do not retain the GILZ form missing the N-terminal region ( ⁇ N-GILZ).
  • TSC protein family comprises leucine zipper proteins (such as GILZ, TSC-22, THG-1, DIP) sharing an evolutionary conserved dimerization domain comprised between less conserved N-terminal and C-terminal domains. These latter ones are probably the regions characterising the functions of TSC proteins, meanwhile the conserved one allow the homodimerization and, possibly, heterodimerization of these proteins.
  • leucine zipper proteins such as GILZ, TSC-22, THG-1, DIP
  • TSC-related protein whose structure has been solved is porcine DIP (Seidel G et al., 1997), a 77-residue long protein which is highly homologous to the segment 58-134 of human GILZ, corresponding to central / C-terminal portion of GILZ and TSC-22. Therefore, no structure data are available for most of the area delimited by the previous experiments as the Raf binding domain of GILZ.
  • this N-terminal region comprises an helical region internal to a random coiled area (residues 1-20), an area in which extended strands are strongly predicted (residues 21-50), and a long helical structure including the four key leucine residues (at positions 76, 83, 90 and 97) and an asparagine residue (at position 87) within the leucine zipper domain, which are compatible with the canonical leucine zipper structure of the family ( figure 15 ).
  • the GILZ sequence allowing the Raf binding is contained in GILZ(16-36), as shown in figure 14 .
  • GILZ binding determinants structural features may be present in the most N-terminal and C-terminal sequences of this fragment, since the central region is highly hydrophobic (see the sequence STSFFSSLL between 21 and 29).
  • polypeptides or peptides comprising at least 5 consecutive amino acids of SEQ ID NO: 3 are fragments of the N-terminal domain of GILZ corresponding to structural elements of such region.
  • these peptides correspond to the sequences GILZ(1-20), GILZ(21-50), GILZ (1-50), GILZ (10-30), GILZ (10-40), GILZ (16-22), GILZ (30-36), GILZ (10-50), GILZ (30-50), GILZ(16-58), or GILZ(1-36).
  • GILZ fragments including, partly or completely, sequences belonging to one or more of these structural elements can be usefully tested. Such fragments should display the same novel biological activity of GILZ characterized in the present invention, as determined by means of routine experimentation comprising subjecting such an analog to the assays disclosed in the present application.
  • results obtained with GILZ mutants indicate that the N-terminal domain of GILZ interacts with the Ras binding domain of Raf. Moreover, the results obtained using non-dimerising GILZ mutants also indicate that, different from GILZ/NF-kB interaction, GILZ/Raf interaction does not require GILZ dimerisation and that it is compatible with a 1:1, protein to protein, interaction model. Compared with the evidences on the GILZ interaction with NF-kB, it can be suggested that different molecular portions are responsible for the interaction with NF-kB and Raf and that, possibly, GILZ could preferentially bind to NF-kB or Raf depending on the ratio of its dimeric and/or monomeric arrangement.
  • GILZ may bind Raf alone or together with other Raf interacting proteins (such Ras, as demonstrated in example 2). depending on the cell state (un-/stimulated, un/transformed) and/or on the cell type (peripheral/ central lymphocyte, or other tissues)

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Claims (27)

  1. Peptide capable de se lier à une protéine Raf et d'inhiber la voie des MAPK, choisi parmi :
    a) un peptide qui consiste en la séquence des acides aminés n° 1 à 50 de la Séquence N° 1 ou de la Séquence N° 2 ;
    b) un fragment d'un peptide défini en (a), comportant au moins 10 résidus d'acides aminés, et de préférence, au moins 20 résidus d'acides aminés ;
    c) et un fragment défini en (b), doté d'une séquence correspondant aux résidus d'acides aminés n° 1 à 20, n° 21 à 50, n° 10 à 30 ou n° 10 à 50 de la Séquence N° 1 ou de la Séquence N° 2.
  2. Peptide capable de se lier à une protéine Raf et d'inhiber la voie des MAPK, choisi parmi :
    a) un peptide qui consiste en la séquence d'acides aminés donnée en tant que Séquence N° 3 ;
    b) un fragment d'un peptide défini en (a), comportant au moins 5 résidus consécutifs d'acides aminés de la Séquence N° 3 ;
    c) et un fragment défini en (b), qui consiste en une séquence correspondant aux résidus d'acides aminés n° 1 à 7 ou n° 15 à 21 de la Séquence N° 3.
  3. Peptide conforme à la revendication 1 ou 2, se présentant sous forme de fractions actives, de précurseurs, de sels ou de dérivés, lesquels dérivés peuvent être préparés à partir des groupes fonctionnels présents sur les chaînes latérales des résidus d'acides aminés ou à partir des groupes amino ou carboxyle terminaux.
  4. Peptide mimétique des peptides conformes à l'une des revendications 1 à 3.
  5. Peptide conforme à l'une des revendications 1 à 4, chimiquement modifié avec des molécules qui, parce qu'elles sont transportées naturellement à travers les membranes cellulaires, en facilitent l'entrée dans les cellules ou en améliorent l'aptitude à traverser les membranes cellulaires et à pénétrer dans le cytoplasme.
  6. Séquence d'ADN codant un peptide dérivé de GILZ, conforme à la revendication 1 ou 2.
  7. Vecteur d'expression comprenant un ADN conforme à la revendication 6, accompagné de n'importe quels éléments appropriés servant au démarrage ou à l'arrêt de la transcription ou de la traduction, ainsi que de n'importe quelle autre séquence supplémentaire.
  8. Peptide, ADN ou vecteur conforme à l'une des revendications 1 à 7, conçu pour servir de médicament.
  9. Composition pharmaceutique comprenant un composté conforme à l'une des revendications 1 à 8, en tant qu'ingrédient actif, ainsi qu'un véhicule, excipient, stabilisant ou diluant pharmacologiquement admissible.
  10. Cellule hôte transformée à l'aide d'un vecteur d'expression conforme à la revendication 7.
  11. Préparation purifiée d'un peptide conforme à l'une des revendications 1 à 5, contenant au moins 1 % de ce composé.
  12. Procédé de criblage in vitro de composés susceptibles de se lier à une protéine Raf et d'inhiber la voie des MAPK, dans lequel procédé l'on compare l'effet de ces composés à l'effet de peptides conformes à l'une des revendications 1 à 3.
  13. Trousse conçue pour un criblage in vitro ou in vivo de composés susceptibles de se lier à une protéine Raf et d'inhiber la voie des MAPK, criblage effectué par comparaison de l'effet de ces composés à l'effet de peptides conformes à l'une des revendications 1 à 3.
  14. Procédé permettant d'inhiber, chez des cellules en culture, une prolifération cellulaire médiée par MAPK indésirable, dans lequel procédé l'on met lesdites cellules en présence d'une quantité efficace d'un peptide conforme à l'une des revendications 1 à 5.
  15. Emploi d'un peptide, ADN ou vecteur, conforme à l'une des revendications 1 à 7, en vue de la fabrication d'un médicament conçu pour le traitement d'un cancer.
  16. Emploi conforme à la revendication 15, le cancer étant choisi parmi les suivants : carcinomes, tumeurs hématopoïétiques de lignée lymphoïde ou myéloïde, lymphomes, leucémie lymphocytaire, tumeurs d'origine mésenchymateuse et mélanome.
  17. Emploi d'un peptide, ADN ou vecteur, conforme à l'une des revendications 1 à 7, en vue de la fabrication d'un médicament conçu pour le traitement d'une maladie auto-immune ou d'une maladie inflammatoire.
  18. Emploi conforme à la revendication 17, la maladie auto-immune ou la maladie inflammatoire étant choisie parmi les suivantes : lupus érythémateux aigu disséminé (LEAD), diabète de type 1, vasculite, hépatite chronique active auto-immune, colite ulcéreuse, maladie de Crohn, maladies allergiques, syndrome néphrotique, sarcoïdose et polyarthrite rhumatoïde.
  19. Emploi d'un peptide, ADN ou vecteur, conforme à l'une des revendications 1 à 7, en vue de la fabrication d'un médicament conçu pour prévenir le rejet d'un organe transplanté, chez des receveurs d'une allogreffe ou d'une xénogreffe.
  20. Peptide consistant en la séquence d'acides aminés donnée en tant que Séquence N° 1 ou Séquence N° 2, conçu pour servir de médicament.
  21. Procédé de criblage in vitro de composés susceptibles de se lier à une protéine Raf et d'inhiber la voie des MAPK, dans lequel procédé l'on compare l'effet de ces composés à l'effet d'un peptide consistant en la séquence d'acides aminés donnée en tant que Séquence N° 1 ou Séquence N° 2.
  22. Trousse conçue pour un criblage in vitro ou in vivo de composés susceptibles de se lier à une protéine Raf et d'inhiber la voie des MAPK, criblage effectué par comparaison de l'effet de ces composés à l'effet d'un peptide consistant en la séquence d'acides aminés donnée en tant que Séquence N° 1 ou Séquence N° 2.
  23. Emploi d'un peptide consistant en la séquence d'acides aminés donnée en tant que Séquence N° 1 ou Séquence N° 2, en vue de la fabrication d'un médicament conçu pour le traitement d'un cancer.
  24. Emploi conforme à la revendication 23, le cancer étant choisi parmi les suivants : carcinomes, tumeurs hématopoïétiques de lignée lymphoïde ou myéloïde, lymphomes, leucémie lymphocytaire, tumeurs d'origine mésenchymateuse et mélanome.
  25. Emploi d'un peptide consistant en la séquence d'acides aminés donnée en tant que Séquence N° 1 ou Séquence N° 2, en vue de la fabrication d'un médicament conçu pour le traitement d'une maladie auto-immune ou d'une maladie inflammatoire.
  26. Emploi conforme à la revendication 25, la maladie auto-immune ou la maladie inflammatoire étant choisie parmi les suivantes : lupus érythémateux aigu disséminé (LEAD), diabète de type 1, vasculite, hépatite chronique active auto-immune, colite ulcéreuse, maladie de Crohn, maladies allergiques, syndrome néphrotique, sarcoïdose et polyarthrite rhumatoïde.
  27. Emploi d'un peptide consistant en la séquence d'acides aminés donnée en tant que Séquence N° 1 ou Séquence N° 2, en vue de la fabrication d'un médicament conçu pour prévenir le rejet d'un organe transplanté, chez des receveurs d'une allogreffe ou d'une xénogreffe.
EP02793099A 2001-12-21 2002-12-20 Composes de liaison raf/ras Expired - Lifetime EP1465996B1 (fr)

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EP01000788 2001-12-21
EP01000788 2001-12-21
EP02793099A EP1465996B1 (fr) 2001-12-21 2002-12-20 Composes de liaison raf/ras
PCT/EP2002/014663 WO2003054193A2 (fr) 2001-12-21 2002-12-20 Nouveaux composes de liaison raf/ras

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JP (1) JP2005525092A (fr)
AT (1) ATE413453T1 (fr)
AU (1) AU2002358780B2 (fr)
CA (1) CA2469263A1 (fr)
DE (1) DE60229781D1 (fr)
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HUP0401127A3 (en) * 2001-05-02 2006-03-28 Purdue Research Foundation Treatment and diagnosis of macrophage disease
JP2007526238A (ja) * 2003-05-06 2007-09-13 パーデュー・リサーチ・ファウンデーション マクロファージまたは葉酸受容体を標的とする狼瘡治療法
FR2875136B1 (fr) 2004-09-10 2007-12-14 Assist Publ Hopitaux De Paris Utilisation de la proteine gilz exprimee dans les cellules dendritiques pour moduler la reponse immune specifique d'un antigene
EP1904183B1 (fr) 2005-07-05 2014-10-15 Purdue Research Foundation Composition pharmaceutique pour le traitement de l'arthrose
WO2014172639A1 (fr) * 2013-04-19 2014-10-23 Ruga Corporation Inhibiteurs des kinases raf
KR20200109325A (ko) * 2018-01-18 2020-09-22 프레드 헛친슨 켄서 리서치 센터 세포 활성화 상태를 조절함으로써 생체내 면역 세포의 염증 상태의 변경
US11524091B2 (en) * 2018-08-21 2022-12-13 Augusta University Research Institute, Inc. GILZ formulations for wound healing

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EP0884385A1 (fr) * 1997-04-28 1998-12-16 Applied Research Systems Ars Holding N.V. Modulateurs intracellulaires des voies de mort cellulaire apoptopique
US6867283B2 (en) * 2001-05-16 2005-03-15 Technion Research & Development Foundation Ltd. Peptides capable of binding to MHC molecules, cells presenting such peptides, and pharmaceutical compositions comprising such peptides and/or cells

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WO2003054193A2 (fr) 2003-07-03
WO2003054193A3 (fr) 2003-10-23
DE60229781D1 (de) 2008-12-18
EP1465996A2 (fr) 2004-10-13
AU2002358780B2 (en) 2008-03-06
CA2469263A1 (fr) 2003-07-03
AU2002358780A1 (en) 2003-07-09
ATE413453T1 (de) 2008-11-15
US20050164906A1 (en) 2005-07-28
JP2005525092A (ja) 2005-08-25
IL162601A0 (en) 2005-11-20

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